Diagnostic Microbiology and Infectious Disease 81 (2015) 1–3
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Bacteriology
Molecular epidemiology of methicillin-resistant Staphylococcus aureus from 4 Cuban hospitals Osney Leiva Peláez a,b,1, Miloš Stojanov a,1, Angela Mariana Zayas Tamayo b, Grether Barreras García b, Mabel González Aleman c, Leonor Martínez Ceballos d, Jorge Luis Muñoz del Campo e, Olga Bello Rodríguez f, Leonora Gonzalez Mesa g, Dominique S. Blanc a,h,⁎ a
Service of Hospital Preventive Medicine, Lausanne University Hospital, Switzerland Microbiology Department, National Center for Scientific Investigation (CNIC), Havana, Cuba c Infectiology Department, William Soler Hospital, Havana, Cuba d Microbiology Department, Enrique Cabrera Hospital, Havana, Cuba e Microbiology Department, Leonor Pérez Hospital, Havana, Cuba f Microbiology Department, Center for Medical Surgical Investigations (CIMEQ), Havana, Cuba g Molecular Biology Department, Cuban Neuroscience Center, Havana, Cuba h Institute of Microbiology, Lausanne University Hospital, Switzerland b
a r t i c l e
i n f o
Article history: Received 23 July 2014 Received in revised form 23 October 2014 Accepted 27 October 2014 Available online 4 November 2014
a b s t r a c t During a 1-year period, 87 methicillin-resistant Staphylococcus aureus isolates were collected from 4 major Cuban hospitals for epidemiological analysis. The majority (86%) were related to the community-associated USA300 clone, whereas the remaining belonged to a new clone ST72-V. Interestingly, no hospital-associated clone was found in these Cuban hospitals. © 2015 Elsevier Inc. All rights reserved.
Keywords: MRSA Cuba ST8 ST72 Molecular epidemiology
Since its emergence in the early 1960s, methicillin-resistant Staphylococcus aureus (MRSA) has been 1 of the most important microorganisms associated with human health. This situation led to national and international studies, focusing on antibiotic resistance, surveillance, and molecular characterization of MRSA clones, aiming to control the burden of MRSA infections. In Latin America and the Caribbean, MRSA is a common cause of nosocomial infections and increasingly associated with communityacquired strains (CA-MRSA) (Garza-Gonzalez and Dowzicky, 2013; Guzman-Blanco et al., 2009). Several international surveillance programs on the antimicrobial susceptibility of bacterial pathogens, such as the SENTRY Antimicrobial Surveillance Program or the Tigecycline Evaluation and Surveillance Trial, have concentrated on these regions (Garza-Gonzalez and Dowzicky, 2013; Sader et al., 2004). A recent study, done in Latin American hospitals, reported a rate of 48% of MRSA among 921 S. aureus isolates. Intercountry differences ranged from 29% in Brazil and Colombia to 79% in Peru (Jones et al.,
⁎ Corresponding author. Tel.: +41-21-314-02-59. E-mail address: [email protected] (D.S. Blanc). 1 Shared first authorship. http://dx.doi.org/10.1016/j.diagmicrobio.2014.10.012 0732-8893/© 2015 Elsevier Inc. All rights reserved.
2013). In addition, several studies have described the molecular epidemiology of MRSA in Latin America (Guzman-Blanco et al., 2009; Rodriguez-Noriega et al., 2010; Reyes et al., 2009; Uhlemann et al., 2012). Cuba, however, did not participate in these international surveillance studies, and data regarding MRSA prevalence in the country are scarce. Furthermore, very little is known about the molecular epidemiology of MRSA in Cuba, primarily due to limited resources and technological expertise. A single molecular epidemiological report on Cuban MRSA indicated that among 40 clinical strains, the majority belonged to spa-type t149, previously associated with nosocomial infections (Hopman et al., 2012). In addition, some strains were related to the USA300 CA-MRSA clone (Tenover et al., 2006; Nimmo, 2012). However, a detailed analysis of the genetic characteristics of these strains, including the presence of virulence and antibiotic resistance genes, SCCmec type, presence of PVL, or arginine catabolic mobile element (ACME), was not performed. In the present study, MRSA isolates recovered from 4 Cuban hospitals were characterized by a variety of phenotypic and genotypic methods including microarrays to detect virulence and antibiotic resistance genes. From January to December 2011, 87 MRSA isolates were collected on a voluntary basis from 4 major hospitals in the Havana City
2
O. Leiva Peláez et al. / Diagnostic Microbiology and Infectious Disease 81 (2015) 1–3
Table 1 Demographics, sites of isolation and antibiotic resistance patterns of the 87 Cuban MRSA isolates.
Hospitals Leonor Perez Paediatric Hospital William Soler Paediatric Hospital Center for Medical Surgical investigations (CIMEQ) Enrique Cabrera University Hospital Patients Gender Female Male Age Mean Age groups 0–17 18–50 51–76 Site of infection Skin lesion Surgical wound Blood Bronchial secretion Others Antibiotic resistance profilesa P, FOX, E, CIP P, FOX, CIP, GM, E, CC P, FOX, GM P, FOX, CIP P, FOX, GM, CIP P, FOX, GM, E, CIP
ST8-IV
ST72-V
Total
25 (93%) 18 (69%) 23 (92%)
2 (7%) 8 (31%) 2 (8%)
27 (31%) 26 (30%) 25 (29%)
9 (100%)
9 (10%)
34 (45%) 41 (55%)
7 (58%) 5 (42%)
41 (47%) 46 (53%)
22.96
20.58
23
41 (55%) 27 (36%) 7 (9%)
10 (84%) 1 (8%) 1 (8%)
51 (59%) 28 (32%) 8 (9%)
40 (53%) 19 (26%) 4 (5%) 4 (5%) 8 (11%)
10 (84%) 1 (8%) 1 (8%)
50 (57%) 20 (23%) 5 (6%) 4 (5%) 8 (9%)
10 (13%) 2 (3%) 41 (55%) 5 (7%) 17 (22%) 12 (100%)
41 (47%) 17 (20%) 12 (14%) 10 (11%) 5 (6%) 2 (2%)
a
Tested antibiotics: P = penicillin; FOX = cefoxitin; E = erythromycin; CIP = ciprofloxacin; GM = gentamicin; CC = clindamycin; fusidic acid; mupirocin; rifampicin; cotrimoxazole; vancomycin.
Province in Cuba (William Soler Paediatric Hospital, Enrique Cabrera University Hospital, Leonor Perez Paediatric Hospital and Center for Medical Surgical Investigations [CIMEQ]). The origin of isolates, sex, and age of patients are summarized in Table 1. The isolates were confirmed as S. aureus by standard identification tests (coagulase and protein A agglutination, catalase, and DNAse tests). Susceptibility to 11 antibiotics, selected for typing purposes (Blanc et al., 1994, 1996), was assessed using the Kirby–Bauer method according to EUCAST guidelines (http://www.eucast.org/). All strains were resistant to cefoxitin, and approximately one fourth were multidrug resistant (i.e., resistant to betalactams with at least 3 other classes of antibiotics, Table 1). All strains were susceptible to cotrimoxazole, fusidic acid, mupirocin, rifampicin, and vancomycin. Molecular typing was performed on each isolate using double locus sequence typing (DLST) as previously described (Kuhn et al., 2007) (www.dlst.org). Multilocus sequence typing (MLST) (Enright et al., 2000) was performed on 1 isolate of every DLST type. The isolates were additionally characterized by spa typing (Frenay et al., 1996), SCCmec typing according to the method of Kondo et al. (2007), PCR
Table 2 Molecular characterization the 87 Cuban MRSA isolates. No of MLST DLST isolates
Spatype
SCCmec PVL ACME dru type
59
ST8
t008
IVa
pos
pos
12 1 3 11
ST8 ST8 ST8 ST72
t008 t008 t211 t13567
IVa IVa IVa V
pos neg pos neg
neg pos pos neg
dt7ag, dt7d, dt9g, dt8w dt7d dt7d dt6w dt11a
1
ST72
pos
neg
dt11a
3-3, 867-3, 877-3, 888-3, 893-3 3-3 3-3 3-6 584-759, 897759 584-759
t13567 V
for the Panton-Valentine leukocidin (PVL) genes, ACME (Diep et al., 2006; Lina et al., 1999), and mec-associated direct repeat unit (dru) typing (Goering et al., 2008). Molecular characteristics of the strains are listed in Table 2. All strains belonged to CC8 and were either ST8 (86.2%) or ST72 (13.7%). ST8 strains carried SCCmec IVa and the majority (83%) were positive for both PVL and ACME, strongly supporting the hypothesis that they were of the CA-MRSA USA300 strain type. Eight different DLST profiles, 4 different dru types, and differences in antimicrobial susceptibility patterns were seen in these ST8 strains (Table 2). This diversity and the lack of a predominant pattern suggest that the USA300 clone may not have recently spread in Cuba or that it was introduced on multiple occasions. The overall mean age of patients carrying this clone was 23 years (Table 1), which is in agreement with other studies associating USA300-related infections with children and young patients (Crum et al., 2006; Saunders et al., 2007). Interestingly, none of the isolates were the USA300-LV variant (SCCmec-IVc and ACME negative) predominantly found in Latin American countries (Reyes et al., 2009; Nimmo, 2012; Garcia et al., 2011). All ST72 isolates carried SCCmec V and belonged to novel spa and DLST types, t13567, and 584-759, respectively (Table 2). None of them carried the ACME, and only 1 was positive for PVL. The lower diversity of DLST, dru types, and antimicrobial susceptibility patterns suggest that ST72 might be a newly spreading clone in these hospitals. The absence of this strain in the previous study characterizing Cuban MRSA isolated in 2008 (Hopman et al., 2012) supports this hypothesis. This clone carried SCCmec V and was associated with younger patients (mean age = 22 years) suggesting CA-MRSA. However, more definitive assessment of hospital or community association would require additional supplementary epidemiological data. Nine isolates, selected according to their DLST type, susceptibility patterns, hospital origin, and other genetic characteristics, were subjected to DNA microarray analysis using the StaphyType kit (Alere Technologies GmbH, Jena, Germany) (Monecke et al., 2008). Results confirmed MLST typing and presence/absence of antibiotic resistance genes correlated with the variability of the phenotypic antimicrobial susceptibility patterns within the same clone (Table S1). While all strains included in the study originated from hospitalized patients, it is remarkable that none were associated with known hospital-acquired MRSA clones (Deurenberg and Stobberingh, 2008; Stefani et al., 2012). Instead, the majority of patients carried ST8-IV, PVL-, and ACME-positive strains, whose characteristics are typical of the USA300 clone. This clone (spa-type t008) was already found in a previous study characterizing Cuban MRSA isolated in 2008 (Hopman et al., 2012), together with spa-types t037 and t149, the latter being the most predominant at that time. Our data suggest the expansion of the USA300-related clone during the last years, which may represent a serious problem in Cuban hospitals, together with the emergence of a new clone (ST72-V). A limitation of our study is the representativeness of the collection of isolates that was performed on a voluntary basis. Due to lack of microbiological data and scarce surveillance programs in this country, information on MRSA rates in hospitals was unavailable. It is probable that more patients were colonized/infected with MRSA in these hospitals; however, we had no indication to suspect bias in the selection of isolates. In conclusion, we found that, in 4 major hospitals in the Havana region, only 2 clones accounted for MRSA infections during a 1-year period. One was a novel ST72-V clone, but the majority of isolates were genetically related to the CA-MRSA USA300 clone. To our knowledge, this is the first report characterizing in detail MRSA clones in Cuba.
Transparency declaration There are no conflicting interests. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2014.10.012.
O. Leiva Peláez et al. / Diagnostic Microbiology and Infectious Disease 81 (2015) 1–3
Acknowledgments We are grateful to Caroline Choulat for the technical assistance and Richard Goering for the helpful discussion correcting the manuscript. OLP was supported by the Swiss Government Excellence Scholarships for Foreign Scholars. Ethics approval was not required for this study.
References Garza-Gonzalez E, Dowzicky MJ. Changes in Staphylococcus aureus susceptibility across Latin America between 2004 and 2010. Braz J Infect Dis 2013;17:13–9. Guzman-Blanco M, Mejia C, Isturiz R, Alvarez C, Bavestrello L, Gotuzzo E, et al. Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Latin America. Int J Antimicrob Agents 2009;34:304–8. Sader HS, Jones RN, Gales AC, Silva JB, Pignatari AC, Group SP. Sentry antimicrobial surveillance program report: Latin American and Brazilian results for 1997 through 2001. Braz J Infect Dis 2004;8:25–79. Jones RN, Guzman-Blanco M, Gales AC, Gallegos B, Castro AL, Martino MD, et al. Susceptibility rates in Latin American nations: report from a regional resistance surveillance program (2011). Braz J Infect Dis 2013;17:672–81. Rodriguez-Noriega E, Seas C, Guzman-Blanco M, Mejia C, Alvarez C, Bavestrello L, et al. Evolution of methicillin-resistant Staphylococcus aureus clones in Latin America. Int J Infect Dis 2010;14:e560–6. Reyes J, Rincon S, Diaz L, Panesso D, Contreras GA, Zurita J, et al. Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America. Clin Infect Dis 2009;49:1861–7. Uhlemann AC, Dumortier C, Hafer C, Taylor BS, Sanchez J, Rodriguez-Taveras C, et al. Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones. Eur J Clin Microbiol Infect Dis 2012;31: 505–11. Hopman J, Peraza GT, Espinosa F, Klaassen CH, Velazquez DM, Meis JF, et al. USA300 methicillin-resistant Staphylococcus aureus in Cuba. Antimicrob Resist Infect Control 2012;1:2. Tenover FC, McDougal LK, Goering RV, Killgore G, Projan SJ, Patel JB, et al. Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States. J Clin Microbiol 2006;44:108–18. Nimmo GR. USA300 abroad: global spread of a virulent strain of community-associated methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 2012;18:725–34. Blanc DS, Lugeon C, Wenger A, Siegrist HH, Francioli P. Quantitative antibiogram typing using inhibition zone diameters compared with ribotyping for epidemiological typing of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 1994;32:2505–9.
3
Blanc DS, Petignat C, Moreillon P, Wenger A, Bille J, Francioli P. Quantitative antibiogram as a typing method for the prospective epidemiological surveillance and control of MRSA: comparison with molecular typing. Infect Control Hosp Epidemiol 1996;17: 654–9. Kuhn G, Francioli P, Blanc DS. Double-locus sequence typing using clfb and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2007;45:54–62. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38:1008–15. Frenay HM, Bunschoten AE, Schouls LM, van Leeuwen WJ, Vandenbroucke-Grauls CM, Verhoef J, et al. Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. Eur J Clin Microbiol Infect Dis 1996;15: 60–4. Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, et al. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother 2007;51:264–74. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, et al. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet 2006;367:731–9. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999;29:1128–32. Goering RV, Morrison D, Al-Doori Z, Edwards GF, Gemmell CG. Usefulness of mecassociated direct repeat unit (dru) typing in the epidemiological analysis of highly clonal methicillin-resistant Staphylococcus aureus in Scotland. Clin Microbiol Infect 2008;14:964–9. Crum NF, Lee RU, Thornton SA, Stine OC, Wallace MR, Barrozo C, et al. Fifteen-year study of the changing epidemiology of methicillin-resistant Staphylococcus aureus. Am J Med 2006;119:943–51. Saunders A, Panaro L, McGeer A, Rosenthal A, White D, Willey BM, et al. A nosocomial outbreak of community-associated methicillin-resistant Staphylococcus aureus among healthy newborns and postpartum mothers. Can J Infect Dis Med Microbiol 2007; 18:128–32. Garcia C, Deplano A, Denis O, Leon M, Siu H, Chincha O, et al. Spread of community-associated methicillin-resistant Staphylococcus aureus to Peru. J Infect 2011;63:482–3. Monecke S, Jatzwauk L, Weber S, Slickers P, Ehricht R. DNA microarray-based genotyping of methicillin-resistant Staphylococcus aureus strains from Eastern Saxony. Clin Microbiol Infect 2008;14:534–45. Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus. Infect Genet Evol 2008;8:747–63. Stefani S, Chung DR, Lindsay JA, Friedrich AW, Kearns AM, Westh H, et al. Meticillinresistant Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods. Int J Antimicrob Agents 2012;39:273–82.
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Bacteriology
Molecular epidemiology of methicillin-resistant Staphylococcus aureus from 4 Cuban hospitals Osney Leiva Peláez a,b,1, Miloš Stojanov a,1, Angela Mariana Zayas Tamayo b, Grether Barreras García b, Mabel González Aleman c, Leonor Martínez Ceballos d, Jorge Luis Muñoz del Campo e, Olga Bello Rodríguez f, Leonora Gonzalez Mesa g, Dominique S. Blanc a,h,⁎ a
Service of Hospital Preventive Medicine, Lausanne University Hospital, Switzerland Microbiology Department, National Center for Scientific Investigation (CNIC), Havana, Cuba c Infectiology Department, William Soler Hospital, Havana, Cuba d Microbiology Department, Enrique Cabrera Hospital, Havana, Cuba e Microbiology Department, Leonor Pérez Hospital, Havana, Cuba f Microbiology Department, Center for Medical Surgical Investigations (CIMEQ), Havana, Cuba g Molecular Biology Department, Cuban Neuroscience Center, Havana, Cuba h Institute of Microbiology, Lausanne University Hospital, Switzerland b
a r t i c l e
i n f o
Article history: Received 23 July 2014 Received in revised form 23 October 2014 Accepted 27 October 2014 Available online 4 November 2014
a b s t r a c t During a 1-year period, 87 methicillin-resistant Staphylococcus aureus isolates were collected from 4 major Cuban hospitals for epidemiological analysis. The majority (86%) were related to the community-associated USA300 clone, whereas the remaining belonged to a new clone ST72-V. Interestingly, no hospital-associated clone was found in these Cuban hospitals. © 2015 Elsevier Inc. All rights reserved.
Keywords: MRSA Cuba ST8 ST72 Molecular epidemiology
Since its emergence in the early 1960s, methicillin-resistant Staphylococcus aureus (MRSA) has been 1 of the most important microorganisms associated with human health. This situation led to national and international studies, focusing on antibiotic resistance, surveillance, and molecular characterization of MRSA clones, aiming to control the burden of MRSA infections. In Latin America and the Caribbean, MRSA is a common cause of nosocomial infections and increasingly associated with communityacquired strains (CA-MRSA) (Garza-Gonzalez and Dowzicky, 2013; Guzman-Blanco et al., 2009). Several international surveillance programs on the antimicrobial susceptibility of bacterial pathogens, such as the SENTRY Antimicrobial Surveillance Program or the Tigecycline Evaluation and Surveillance Trial, have concentrated on these regions (Garza-Gonzalez and Dowzicky, 2013; Sader et al., 2004). A recent study, done in Latin American hospitals, reported a rate of 48% of MRSA among 921 S. aureus isolates. Intercountry differences ranged from 29% in Brazil and Colombia to 79% in Peru (Jones et al.,
⁎ Corresponding author. Tel.: +41-21-314-02-59. E-mail address: [email protected] (D.S. Blanc). 1 Shared first authorship. http://dx.doi.org/10.1016/j.diagmicrobio.2014.10.012 0732-8893/© 2015 Elsevier Inc. All rights reserved.
2013). In addition, several studies have described the molecular epidemiology of MRSA in Latin America (Guzman-Blanco et al., 2009; Rodriguez-Noriega et al., 2010; Reyes et al., 2009; Uhlemann et al., 2012). Cuba, however, did not participate in these international surveillance studies, and data regarding MRSA prevalence in the country are scarce. Furthermore, very little is known about the molecular epidemiology of MRSA in Cuba, primarily due to limited resources and technological expertise. A single molecular epidemiological report on Cuban MRSA indicated that among 40 clinical strains, the majority belonged to spa-type t149, previously associated with nosocomial infections (Hopman et al., 2012). In addition, some strains were related to the USA300 CA-MRSA clone (Tenover et al., 2006; Nimmo, 2012). However, a detailed analysis of the genetic characteristics of these strains, including the presence of virulence and antibiotic resistance genes, SCCmec type, presence of PVL, or arginine catabolic mobile element (ACME), was not performed. In the present study, MRSA isolates recovered from 4 Cuban hospitals were characterized by a variety of phenotypic and genotypic methods including microarrays to detect virulence and antibiotic resistance genes. From January to December 2011, 87 MRSA isolates were collected on a voluntary basis from 4 major hospitals in the Havana City
2
O. Leiva Peláez et al. / Diagnostic Microbiology and Infectious Disease 81 (2015) 1–3
Table 1 Demographics, sites of isolation and antibiotic resistance patterns of the 87 Cuban MRSA isolates.
Hospitals Leonor Perez Paediatric Hospital William Soler Paediatric Hospital Center for Medical Surgical investigations (CIMEQ) Enrique Cabrera University Hospital Patients Gender Female Male Age Mean Age groups 0–17 18–50 51–76 Site of infection Skin lesion Surgical wound Blood Bronchial secretion Others Antibiotic resistance profilesa P, FOX, E, CIP P, FOX, CIP, GM, E, CC P, FOX, GM P, FOX, CIP P, FOX, GM, CIP P, FOX, GM, E, CIP
ST8-IV
ST72-V
Total
25 (93%) 18 (69%) 23 (92%)
2 (7%) 8 (31%) 2 (8%)
27 (31%) 26 (30%) 25 (29%)
9 (100%)
9 (10%)
34 (45%) 41 (55%)
7 (58%) 5 (42%)
41 (47%) 46 (53%)
22.96
20.58
23
41 (55%) 27 (36%) 7 (9%)
10 (84%) 1 (8%) 1 (8%)
51 (59%) 28 (32%) 8 (9%)
40 (53%) 19 (26%) 4 (5%) 4 (5%) 8 (11%)
10 (84%) 1 (8%) 1 (8%)
50 (57%) 20 (23%) 5 (6%) 4 (5%) 8 (9%)
10 (13%) 2 (3%) 41 (55%) 5 (7%) 17 (22%) 12 (100%)
41 (47%) 17 (20%) 12 (14%) 10 (11%) 5 (6%) 2 (2%)
a
Tested antibiotics: P = penicillin; FOX = cefoxitin; E = erythromycin; CIP = ciprofloxacin; GM = gentamicin; CC = clindamycin; fusidic acid; mupirocin; rifampicin; cotrimoxazole; vancomycin.
Province in Cuba (William Soler Paediatric Hospital, Enrique Cabrera University Hospital, Leonor Perez Paediatric Hospital and Center for Medical Surgical Investigations [CIMEQ]). The origin of isolates, sex, and age of patients are summarized in Table 1. The isolates were confirmed as S. aureus by standard identification tests (coagulase and protein A agglutination, catalase, and DNAse tests). Susceptibility to 11 antibiotics, selected for typing purposes (Blanc et al., 1994, 1996), was assessed using the Kirby–Bauer method according to EUCAST guidelines (http://www.eucast.org/). All strains were resistant to cefoxitin, and approximately one fourth were multidrug resistant (i.e., resistant to betalactams with at least 3 other classes of antibiotics, Table 1). All strains were susceptible to cotrimoxazole, fusidic acid, mupirocin, rifampicin, and vancomycin. Molecular typing was performed on each isolate using double locus sequence typing (DLST) as previously described (Kuhn et al., 2007) (www.dlst.org). Multilocus sequence typing (MLST) (Enright et al., 2000) was performed on 1 isolate of every DLST type. The isolates were additionally characterized by spa typing (Frenay et al., 1996), SCCmec typing according to the method of Kondo et al. (2007), PCR
Table 2 Molecular characterization the 87 Cuban MRSA isolates. No of MLST DLST isolates
Spatype
SCCmec PVL ACME dru type
59
ST8
t008
IVa
pos
pos
12 1 3 11
ST8 ST8 ST8 ST72
t008 t008 t211 t13567
IVa IVa IVa V
pos neg pos neg
neg pos pos neg
dt7ag, dt7d, dt9g, dt8w dt7d dt7d dt6w dt11a
1
ST72
pos
neg
dt11a
3-3, 867-3, 877-3, 888-3, 893-3 3-3 3-3 3-6 584-759, 897759 584-759
t13567 V
for the Panton-Valentine leukocidin (PVL) genes, ACME (Diep et al., 2006; Lina et al., 1999), and mec-associated direct repeat unit (dru) typing (Goering et al., 2008). Molecular characteristics of the strains are listed in Table 2. All strains belonged to CC8 and were either ST8 (86.2%) or ST72 (13.7%). ST8 strains carried SCCmec IVa and the majority (83%) were positive for both PVL and ACME, strongly supporting the hypothesis that they were of the CA-MRSA USA300 strain type. Eight different DLST profiles, 4 different dru types, and differences in antimicrobial susceptibility patterns were seen in these ST8 strains (Table 2). This diversity and the lack of a predominant pattern suggest that the USA300 clone may not have recently spread in Cuba or that it was introduced on multiple occasions. The overall mean age of patients carrying this clone was 23 years (Table 1), which is in agreement with other studies associating USA300-related infections with children and young patients (Crum et al., 2006; Saunders et al., 2007). Interestingly, none of the isolates were the USA300-LV variant (SCCmec-IVc and ACME negative) predominantly found in Latin American countries (Reyes et al., 2009; Nimmo, 2012; Garcia et al., 2011). All ST72 isolates carried SCCmec V and belonged to novel spa and DLST types, t13567, and 584-759, respectively (Table 2). None of them carried the ACME, and only 1 was positive for PVL. The lower diversity of DLST, dru types, and antimicrobial susceptibility patterns suggest that ST72 might be a newly spreading clone in these hospitals. The absence of this strain in the previous study characterizing Cuban MRSA isolated in 2008 (Hopman et al., 2012) supports this hypothesis. This clone carried SCCmec V and was associated with younger patients (mean age = 22 years) suggesting CA-MRSA. However, more definitive assessment of hospital or community association would require additional supplementary epidemiological data. Nine isolates, selected according to their DLST type, susceptibility patterns, hospital origin, and other genetic characteristics, were subjected to DNA microarray analysis using the StaphyType kit (Alere Technologies GmbH, Jena, Germany) (Monecke et al., 2008). Results confirmed MLST typing and presence/absence of antibiotic resistance genes correlated with the variability of the phenotypic antimicrobial susceptibility patterns within the same clone (Table S1). While all strains included in the study originated from hospitalized patients, it is remarkable that none were associated with known hospital-acquired MRSA clones (Deurenberg and Stobberingh, 2008; Stefani et al., 2012). Instead, the majority of patients carried ST8-IV, PVL-, and ACME-positive strains, whose characteristics are typical of the USA300 clone. This clone (spa-type t008) was already found in a previous study characterizing Cuban MRSA isolated in 2008 (Hopman et al., 2012), together with spa-types t037 and t149, the latter being the most predominant at that time. Our data suggest the expansion of the USA300-related clone during the last years, which may represent a serious problem in Cuban hospitals, together with the emergence of a new clone (ST72-V). A limitation of our study is the representativeness of the collection of isolates that was performed on a voluntary basis. Due to lack of microbiological data and scarce surveillance programs in this country, information on MRSA rates in hospitals was unavailable. It is probable that more patients were colonized/infected with MRSA in these hospitals; however, we had no indication to suspect bias in the selection of isolates. In conclusion, we found that, in 4 major hospitals in the Havana region, only 2 clones accounted for MRSA infections during a 1-year period. One was a novel ST72-V clone, but the majority of isolates were genetically related to the CA-MRSA USA300 clone. To our knowledge, this is the first report characterizing in detail MRSA clones in Cuba.
Transparency declaration There are no conflicting interests. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2014.10.012.
O. Leiva Peláez et al. / Diagnostic Microbiology and Infectious Disease 81 (2015) 1–3
Acknowledgments We are grateful to Caroline Choulat for the technical assistance and Richard Goering for the helpful discussion correcting the manuscript. OLP was supported by the Swiss Government Excellence Scholarships for Foreign Scholars. Ethics approval was not required for this study.
References Garza-Gonzalez E, Dowzicky MJ. Changes in Staphylococcus aureus susceptibility across Latin America between 2004 and 2010. Braz J Infect Dis 2013;17:13–9. Guzman-Blanco M, Mejia C, Isturiz R, Alvarez C, Bavestrello L, Gotuzzo E, et al. Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Latin America. Int J Antimicrob Agents 2009;34:304–8. Sader HS, Jones RN, Gales AC, Silva JB, Pignatari AC, Group SP. Sentry antimicrobial surveillance program report: Latin American and Brazilian results for 1997 through 2001. Braz J Infect Dis 2004;8:25–79. Jones RN, Guzman-Blanco M, Gales AC, Gallegos B, Castro AL, Martino MD, et al. Susceptibility rates in Latin American nations: report from a regional resistance surveillance program (2011). Braz J Infect Dis 2013;17:672–81. Rodriguez-Noriega E, Seas C, Guzman-Blanco M, Mejia C, Alvarez C, Bavestrello L, et al. Evolution of methicillin-resistant Staphylococcus aureus clones in Latin America. Int J Infect Dis 2010;14:e560–6. Reyes J, Rincon S, Diaz L, Panesso D, Contreras GA, Zurita J, et al. Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America. Clin Infect Dis 2009;49:1861–7. Uhlemann AC, Dumortier C, Hafer C, Taylor BS, Sanchez J, Rodriguez-Taveras C, et al. Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones. Eur J Clin Microbiol Infect Dis 2012;31: 505–11. Hopman J, Peraza GT, Espinosa F, Klaassen CH, Velazquez DM, Meis JF, et al. USA300 methicillin-resistant Staphylococcus aureus in Cuba. Antimicrob Resist Infect Control 2012;1:2. Tenover FC, McDougal LK, Goering RV, Killgore G, Projan SJ, Patel JB, et al. Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States. J Clin Microbiol 2006;44:108–18. Nimmo GR. USA300 abroad: global spread of a virulent strain of community-associated methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 2012;18:725–34. Blanc DS, Lugeon C, Wenger A, Siegrist HH, Francioli P. Quantitative antibiogram typing using inhibition zone diameters compared with ribotyping for epidemiological typing of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 1994;32:2505–9.
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